The development, remodeling, and pathogenesis of many tissues depend in part on mechanical signals. The foundation of mechanotransduction which transforms the mechanical environment experienced by articular cartilage into a biomolecular response will initially be explored through single chondrocyte manipulation. Healthy chondrocytes experience hydrostatic, compressive, tensile, and shear forces that maintain the phenotype and production of neocartilaginous tissue. Abnormal mechanical forces due to single cycle or fatigue loading, have been shown to alter chondrocyte behavior, resulting in pathological matrix synthesis, increased catabolic activity (degradation), and ultimately osteoarthritis (apoptosis). Studies of the biomechanics of single cells originating from the specific cartilage zones are critical for deciphering the transmission of heterogeneous tissue-level forces to the molecular machinery within the cell. A more complete knowledge of individual cellular biomechanics will prioritize the biomechanical factors most critical to stimulating regenerative processes. As there is no consensus as to the mechanical signals that are optimally effective in modulating cell function, much is left to be studied. We recently developed an integrated /micro- particle image velocimetry/optical tweezers (5PIVOT) system toward this goal. This device was designed as a unique tool intended to study cellular mechanics and facilitate the characterization of mechanobiology. The laser-based technologies have been custom-integrated to physically hold cellular or molecular structures concomitant with monitoring fluid and optical force-induced deformations of the structure. The objective of this project is to establish the feasibility of applying an 5PIVOT system for single chondrocyte biomechanics as a precursor to mechanotransduction. This effort will support the Academic Research Enhancement Award (AREA) Program as directed by the following specific aims: 1) to optimize the integration of two optical systems, not previously used in concert, for measuring multiaxial biomechanical properties of single living cells; 2) to apply a sequence of single and multiple axis stresses to individual chondrocytes while measuring the resulting strain response. Successful outcomes from this AREA can then be used to explore the environment most effective in inducing mechanotransduction. Completion of these studies should provide significant insight into the mechanical response of chondrocytes, contribute to the understanding of pathologic cell states and therapeutic approaches for load-bearing tissues, and guide the design of engineered biomaterials which control cellular function [unreadable] [unreadable] [unreadable]